Compositions and methods for regulating autolytic processes in bacteria

ABSTRACT

A nucleic acid sequence required for regulating the autolytic activity of bacteria is provided. Also provided are polypeptides encoded by the gene or mutant gene as well as vector and host cells for expressing these polypeptides. Methods for identifying and using agents which interact with the gene or mutant gene or polypeptides encoded thereby to inhibit bacterial growth and infectivity are also provided.

INTRODUCTION

This application claims the benefit of priority from U.S. provisionalapplication Serial No. 60/273,791, filed on Mar. 6, 2001; Ser. No.60/312,546, filed on Aug. 15, 2001; and Ser. No. 60/329,140, filed onOct. 12, 2001, whose contents are incorporated herein by reference intheir entireties.

This invention was made in the course of research sponsored by theNational Institute of Health (NIH Grant No. RO1-AI37142). The U.S.government may have certain rights in this invention.

BACKGROUND

Staphylococci are hardy and ubiquitous colonizers of human skin andmucous membranes and were among the first human pathogens identified.These bacteria constitute a medically important genera of microbes asthey are known to produce two types of disease, invasive and toxigenic.

Invasive infections are characterized generally by abscess formationaffecting both skin surfaces and deep tissues. In addition,Staphylococcus aureus (S. aureus) is the second leading cause ofbacteremia in cancer patients. Osteomyelitis, septic arthritis, septicthrombophlebitis and acute bacterial endocarditis are also relativelycommon.

There are also at least three clinical conditions resulting from thetoxigenic properties of Staphylococci. The manifestation of thesediseases result from the actions of exotoxins as opposed to tissueinvasion and bacteremia. These conditions include: Staphylococcal foodpoisoning, scalded skin syndrome and toxic shock syndrome.

S. aureus are non-mobile, non-sporulating gram-positive cocci 0.5–1.5 imin diameter, that occur singly and in pairs, short chains, and irregularthree-dimensional clusters. S. aureus can grow over a wide range ofenvironmental conditions, but they grow best at temperatures between 30°C. and 37° C. and at a neutral pH. They are resistant to desiccation andto chemical disinfection, and they tolerate NaCl concentrations up to12%. It has been found that the growth of the S. aureus becomesunusually sensitive to a high-NaCl concentration by decreasing the Ca²⁺concentration in growth media allowing for autolysis (Ishikawa,Microbiology and Immunology, 2000: 44(2):97–104).

Humans constitute the major reservoir of the S. aureus bacteria. Thecross sectional carriage rate in adults is 15 to 40 percent. The mucousmembranes of the anterior nasopharynx are the principal site ofcarriage. Other sites include the axillae, the vagina, the perineum andoccasionally the gastrointestinal tract. Colonization by S. aureus maybe intermittent or persistent and is probably influenced by bothmicrobial and host factors as well as by the nature of the competingnon-Staphylococcal flora.

The frequency of S. aureus infections has risen dramatically in the past20 years. This has been attributed to the emergence of multipleantibiotic resistant strains and an increasing population of people withweakened immune systems. It is no longer uncommon to isolate S. aureusstrains which are resistant to some or all of the standard antibiotics.This has created a demand for both new anti-microbial agents anddiagnostic tests for this organism. Accordingly, there is a need forbetter understanding of factors which regulate infectivity and growth ofS. aureus.

Genes identified as involved in the infectivity and/or growth of S.aureus include the ArlS regulator, involved in adhesion (Fournier andHooper, Journal of Bacteriology, 2000: 182(14):3955–64), the pbpC gene,which affects the rate of autolysis, (Pinho et al., Journal ofBacteriology, 2000: 182(4):1074–9), lytRS (Brunskill and Bayles, Journalof Bacteriology, 1996: 178(19):5810–2), lrgA and lrgB (Fujimoto et al.,Journal of Bacteriology, 2000: 182(17):4822–8), and lytM identified inautolysis-deficient mutants of S. aureus (Ramadurai and Jayaswal,Journal of Bacteriology, 1997: 179(11):3625–31).

A new genetic locus of S. aureus which regulates autolytic processes hasnow been identified. The gene at this locus, referred to herein as RAT,regulator of autolytic activity, which regulates autolytic processes,has been cloned and sequenced. The phrase, “which regulates expressionof polypeptides involved in autolytic processes” used herein means thatthe gene or polypeptide encoded by the gene controls, modulates orregulates the expression of polypeptides involved in autolytic processessuch as autolytic enzymes (e.g., murein hydrolase, cell wall hydrolase,glycylglycine endopeptidase), polypeptides involved in environmentalsignaling, the secretion of autolysins, or other autolytic processes.Further, it has been shown that a mutation at this site renders S.aureus more susceptible to lysis with antibiotics.

SUMMARY OF THE INVENTION

The present invention provides a new genetic locus of S. aureus andother bacteria that is involved in the control, modulation or regulation(these latter three terms are used as equivalents herein) of autolyticactivity and processes in bacteria. Examples of the gene, a mutant ofthe gene, as well as the polypeptides encoded by the gene and the mutantof the gene are also provided.

An object of the present invention is to provide nucleic acid sequencesisolated from S. aureus and other bacteria which regulate autolyticactivity in bacteria. The nucleic acid sequences referred to herein arethe RAT gene (SEQ ID NO:1), the RAT mutant gene (SEQ ID NO:3) orfragments thereof. In a preferred embodiment, the nucleic acid sequenceis the RAT gene (SEQ ID NO:1).

Another object of the present invention is to provide polypeptidesencoded by the RAT gene and vectors and host cells comprising nucleicacid sequences encoding these polypeptides. In a preferred embodiment,the polypeptide has the sequence of SEQ ID NO:2. The sequence of thepolypeptide encoded by the mutant of the RAT gene is also provided (SEQID NO:4).

Another object of the present invention is to provide a vector whichcomprises a transposon element and a polynucleotide sequence whichencodes the RAT polypeptide (SEQ ID NO:2) or the RAT mutant polypeptide(SEQ ID NO:4) and host cells comprising this vector.

Another object of the present invention is to provide a method ofidentifying agents that modulate autolytic activity of S. aureus andother bacteria through interaction with the RAT gene (SEQ ID NO:1) orRAT mutant gene (SEQ ID NO:3) or polypeptides encoded by RAT or the RATmutant gene. These agents are expected to be useful in the inhibition ofgrowth of S. aureus and other bacteria and in the treatment of hostsinfected by S. aureus and other bacteria. These agents can be used aloneor in combination with an antibiotic such as penicillin to promote lysisof the bacteria.

Accordingly, another object of the present invention is to providemethods for modulating autolytic activity of S. aureus and otherbacteria to inhibit their growth and infectivity by contacting thebacteria with an agent which interacts with the RAT gene (SEQ ID NO:1)or RAT mutant gene (SEQ ID NO:3) or polypeptides encoded thereby.

Yet another object of the present invention is to provide anti-bacterialagents which comprise a compound which prevents or inhibits infectionsby S. aureus or other bacteria via interaction with the RAT gene (SEQ IDNO:1) or RAT mutant gene (SEQ ID NO:3) or polypeptides encoded by RAT orthe RAT mutant gene.

Another object of the present invention is to provide a kit foridentifying the presence of the RAT gene (SEQ ID NO:1) or RAT mutantgene (SEQ ID NO:3) or the polypeptides they encode (SEQ ID NO:2 or SEQID NO:4, respectively).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically demonstrates the effect that subinhibitoryconcentrations of penicillin have on the RAT mutant as compared towild-type.

FIG. 2 graphically represents the viability of the RAT mutant ascompared to wild-type by propidium iodide staining.

DETAILED DESCRIPTION OF THE INVENTION

S. aureus is the most prevalent human pathogen in the Staphylococcalgenus. It remains a major public health concern due to its tenacity,potential destructiveness and increasing resistance to antimicrobialagents. Much research has been focused on identifying genes or geneproducts of S. aureus which serve as targets in the development of newantibacterial agents.

Using transposon mutagenesis, a gene referred to herein as RAT(regulator of autolytic activity)(SEQ ID NO:1), which is specificallyinvolved in regulation of autolytic activity of S. aureus, has beenidentified. To identify the RAT gene, a Tn551 transposon library of S.aureus strain RN6390 was constructed. The library was screened for genesthat affected expression of genes encoding the capsular polysaccharide(cap genes—16 genes encoded within the cap operon) of S. aureus. Usingthe cap promoter linked to the GFP reporter gene (green fluorescentprotein), a mutant was identified that displayed significantly lower cappromoter activity. However, upon growing this mutant, it was discoveredthat this strain grew poorly in 03GL medium, reaching a maximum opticaldensity of 0.8 when the parental strain could achieve an OD650 nm of1.3. The defect was linked to the transposon insertion because thisphenotype could be back-crossed into the parental strain. The region ofthe mutant chromosome where the transposon was inserted was subsequentlysequenced; the sequence of the mutant locus is SEQ ID NO:3. This genewas designated RAT, or regulator of autolytic activity. The RAT mutantgene encodes a protein of 134 residues in length. The RAT mutant is atransposon mutant in which a Tn551 transposon is inserted at the 3′ endof the polynucleotide sequence, yielding a truncated protein orpolypeptide missing the last 13 amino acid residues of the wild-typeprotein. It is believed that the RAT mutant is a partial gene knockoutwhich enables the bacteria to survive. By “knock-out” it is meant thatan alteration in the target gene sequence occurs which results in adecrease of function of the target gene.

Zymogram analysis revealed that the RAT mutant strain displayedsignificantly enhanced autolytic activity as compared with the parent.This defect in autolytic activity was restored upon complementation ofthe mutant with a single copy of the RAT gene inserted into the mutantchromosome. RAT was cloned and sequenced (SEQ ID NO:1). RAT encodes a 17kDa protein of 147 residues in length. Forty-seven of the 147 residues(32%) are charged and the pI of RAT is predicted to be 7.38.

RAT plays a role in regulating autolytic activity of S. aureus. Morespecifically, in the presence of penicillin, the RAT mutant was shown toreadily increase lysis as compared to wild-type S. aureus. Furthermore,inactivation of the RAT locus was shown to render the S. aureus bacteriasensitive to lysis upon growth beyond the mid-log phase. To evaluatewhether the cell lysis of the RAT mutant was additive to the effect of asubinhibitory concentration of penicillin, 200 ng/ml of penicillin wasadded to a growing culture of the RAT mutant at an OD650 nm of 0.5,corresponding to the mid-log phase. Contrary to the wild-type strainthat displayed no increase in optical density (i.e., no growth) uponaddition of penicillin, the RAT mutant exhibited a further reduction inoptical density as the growth cycle progressed (FIG. 1). This finding isconsistent with the additive effect of penicillin upon the lyticpropensity of the RAT mutant late in the growth cycle. Similar resultswere obtained using gentamicin and cephalothin. In plating thesecultures on agar plates without antibiotic selection, it was found thatthe RAT mutant has 1–2 log more kill than the parental strain. Incomparison to the parental strain without antibiotics, the RAT mutantwith subinhibitory concentrations of penicillin has a 3–4 log kill.

To analyze the autolytic activity of the RAT mutant, zymographicanalysis of cell-associated murein hydrolases was performed. Bacterialcells were centrifuged, washed, and resuspended in SDS-gel loadingbuffer, heated for 3 minutes at 100° C., recentrifuged, and thesupernatant applied to a SDS-gel containing heat-killed S. aureusRN4220. Following electrophoresis, the gel was soaked in 0.1 percentTriton X100 at 37° C. overnight to hydrolyze RN4220 cells that had beenattacked by autolytic enzymes in the cell extracts. After incubation,the gel was stained with one percent methylene blue and destained inwater. Clear bands, indicating zones of murein hydrolase activity werefound to be enhanced in the RAT mutant as compared with the wild-typecontrol. As a positive control, a sarA mutant was utilized. The sarAgene normally represses murein hydrolase activity.

To assess the viability of RAT mutant cells late in the growth cycle,the bacterial cells obtained from different parts of the growth cyclewere stained with propidium iodide. Penetration of the cell withpropidium iodide indicates cell death or necrosis. Many of the RATmutant cells picked up the propidium iodide stain as the growth cyclelengthened (FIG. 2), thus accounting for the decrease in opticaldensities in the RAT mutant during the late log phase.

The cell wall morphology of the RAT mutant strain differs from thewild-type strain. RAT mutant cells, undergoing division, exhibited athicker cell wall than the wild-type strain. The outer contour of theRAT mutant was rough while the surface of the wild type cells wassmooth. Irregularities in the outer cell wall have previously beenassociated with altered autolytic activities in mutants. Bacteriumpossessing nucleotide sequences with a sufficient degree of homology toSEQ ID NO:3 also will exhibit an increase sensitivity to lysis.

The bacterial cell wall is maintained by competing enzymes involved inthe synthesis and lysis of the cell wall. Examples of autolytic enzymesinclude glucosamidase, muramidase, amidase, and endopeptidase. Thesynthesis of bacterial cell walls is a dynamic process requiring theprecise regulation of both synthetic and autolytic activities. Theautolytic activity of many bacteria is carefully controlled during thegrowth cycle in particular by regulatory elements. A disruption of theseregulatory elements alters autolytic activity and leads to prematurecell lysis during growth. The RAT mutant has a defect in autolyticactivity which prevents the mutant from reaching the stationary phase ofgrowth. By northern blot analysis, it was shown that the RAT mutationhad an effect on the expression of autolytic enyzmes such as lytN, lytM,and atl. The novel cell wall hydrolase, lytN, and the glycylglycineendopeptidase, lytM, are up-regulated by the RAT mutation. Conversely,the RAT mutation down-regulated the regulators of autolytic activity,namely, lytS, lytR, lrgA, lrgB, arlR, and arlS. Furthermore, the RATmutation affects the expression of certain other S. aureus genes, e.g.,hla, spa, abcA, scdA, pbp2, pbp4, and sspA. Both hla and scdA expressionare down-regulated by the RAT mutation, whereas both spa and abcAexpression are up-regulated by the RAT mutation.

In a comparison of the RAT gene sequence with the genomes of othermicrobes, homologs with significant sequence similarity were identified.The RAT gene or a homolog thereof performs a role in regulating theautolytic activity of bacteria, including but not limited to:Staphylococcus aureus (such as Staphylococcus aureus N315,Staphylococcus aureus strain Mu50, Staphylococcus aureus strain NCTC8325, Staphylococcus aureus MSSA strain, Staphylococcus aureus MRSAstrain, Staphylococcus aureus COL, Staphylococcus epidermidis, andStaphylococcus sciuri), Sinorhizobium species (e.g., meliloti), Listeriaspecies (e.g., monocytogenes),Clostridium species (e.g., acetabutylicum,difficile), Vibrio species (e.g., cholerae), Corynebacterium species(e.g., diptheriae), Brucella species (e.g., suis), Pseudomonas species(e.g., aeruginosa, syringae, putida), Shewanella species (e.g.,putrefasciens), Mesorhizobium species (e.g., loti), Caulobacter species(e.g., crescentus), Lactococcus species (e.g., lactis), Mycobacteriumspecies (e.g., smegmatis, leprae, tuberculosis), Burkholderia species(e.g., mallei, pseudomallei), Geobacter species (e.g., sulfurreducens),Treponema species (e.g., denticola), Bacillus species (e.g.,stearothermophilus, anthracis, subtilis, halodurnas), Escherichiaspecies (e.g., coli), Enterococcus species (e.g., faecalis), Salmonellaspecies (e.g., dublin, enteriditis, paratyphi, typhi), Klebsiellaspecies (e.g., pneumoniae), Bordetella species (e.g., parapertussis),Actinobacillusspecies (e.g., actinomycetemcomitans), Streptomycesspecies (e.g., coelicolor), Streptococcus species (e.g., pyogenes,pneumoniae) and Acinetobacter species.

Useful homologous sequences are those which encode a polypeptide whichincreases bacterial susceptibility to autolysis or increased lysis dueto antibiotic administration. In a preferred embodiment thepolynucleotide sequence is at least 40 percent homologous to the SEQ IDNO:1 or SEQ ID NO:3. In a more preferred embodiment the polynucleotidesequence is at least 60 percent homologous to the SEQ ID NO:1 or SEQ IDNO:3. In a most preferred embodiment the polynucleotide sequence is atleast 80 percent homologous to the SEQ ID NO:1 or SEQ ID NO:3.

The present invention includes the nucleic acid sequences for the RATgene and RAT mutant gene and polypeptides encoded thereby. For purposesof the present invention, polypeptides encoded by the RAT gene and RATmutant gene are referred to herein as RAT polypeptides and RAT mutantpolypeptides, respectively. Exemplary nucleic acid sequences of thepresent invention are SEQ ID NO:1 and SEQ ID NO:3. However, by the term“nucleic acid sequence” it is meant to include any form of DNA or RNAsuch as cDNA or genomic DNA or mRNA, respectively, encoding a RATpolypeptide or RAT mutant polypeptide, or an active fragment thereofwhich are obtained by cloning or produced synthetically by well-knownchemical techniques. DNA may be double- or single-stranded.Single-stranded DNA may comprise the coding or sense strand or thenon-coding or antisense strand. Thus, the term nucleic acid sequencealso includes sequences which hybridize under stringent conditions tothe above-described polynucleotides. As used herein, the term “stringentconditions” means at least 60% homology at hybridization conditions of60° C. at 2×SSC buffer.

In a preferred embodiment, the nucleic acid sequence comprises the cDNAof SEQ ID NO:1or a homologous sequence or fragment thereof which encodesa polypeptide having similar activity to the polypeptide (SEQ ID NO:2)encoded by RAT. In another preferred embodiment, the nucleic acidsequence comprises the cDNA of SEQ ID NO:3 or a homologous sequence orfragment thereof which encodes a polypeptide having similar activity tothe polypeptide (SEQ ID NO:4) encoded by the RAT mutant gene. Due to thedegeneracy of the genetic code, nucleic acid sequences of the presentinvention may also comprise other nucleic acid sequences encoding theRAT polypeptide or RAT mutant polypeptide and derivatives, variants oractive fragments thereof. The present invention also relates to variantsof these nucleic acid sequences which may be naturally occurring, i.e.,allelic variants, or mutants prepared by well known mutagenesistechniques.

The present invention also relates to a conditional mutant whereby theRAT gene or RAT mutant gene can be expressed under an induciblepromoter.

The present invention also relates to vectors comprising nucleic acidsequences of the present invention and host cells which are geneticallyengineered with these vectors to produce active RAT polypeptides or RATmutant polypeptides, or fragments thereof. Generally, any vectorsuitable to maintain, propagate or express the nucleic acid sequences ofthis invention in a host cell may be used for expression in this regard.

The nucleic acid sequences and polypeptides of the present invention, aswell as vectors and host cells expressing the polypeptides are useful asresearch tools to enhance the understanding of the autolytic process ofS. aureus and other bacteria. The methods and compositions of thepresent invention are believed to be effective in other bacteria havingsignificant homology with the polynucleotide sequence of RAT or the RATmutant.

Further, these compositions are useful in the identification of agentswhich interact with either the RAT gene or RAT mutant gene, orpolypeptides encoded thereby to modulate autolytic activity of thebacteria. By “interact” it is meant that the agent increases ordecreases expression of the RAT gene or RAT mutant gene, or increases ordecreases activity of a RAT polypeptide or RAT mutant polypeptide. In apreferred embodiment, agents will decrease, interfere with or inhibitRAT gene or RAT mutant gene expression or decrease, interfere with orinhibit the activity of a RAT polypeptide or RAT mutant polypeptide sothat the bacteria is lysed more easily. Examples of such agents include,but are not limited to, antisense molecules or ribozymes targeted to theRAT gene or RAT mutant gene which inhibit the gene expression, means forintroduction of mutations into the RAT gene or RAT mutant gene whichinhibit gene expression or produce a polypeptide with decreasedactivity, and small organic molecules or peptides which are capable ofinhibiting activity of the RAT polypeptides or RAT mutant polypeptidesor the genes themselves (e.g., by binding to the promoter region of thegene to inhibit transcription and subsequent expression). The activesite of the RAT polypeptide, could be used to simulate activity in themutant. Alternatively, a small compound library can be used to screenfor agents which augment the lytic activity of the RAT gene or RATmutant gene. This augmentation of lytic activity can be monitored bybinding of the small compound to the RAT polypeptide or RAT mutantpolypeptide and determining the ability of the small compound to inducecell lysis.

Accordingly, one aspect of the present invention provides an analoglibrary to produce a very large number of potential molecules forregulating the RAT expression system, and in general the greater thenumber of analogs in the library, the greater the likelihood that atleast one member of the library will effectively regulate the RATexpression system. Designed libraries following a particular templatestructure and limiting amino acid variation at particular positions aremuch preferred, since a single library can encompass all the designedanalogs and the included sequences will be known and presented inroughly equal numbers. By contrast, random substitution at only sixpositions in an amino acid sequence provides over 60 million analogs,which is a library size that begins to present practical limitationseven when utilizing screening techniques as powerful as phage display.Libraries larger than this would pose problems in handling, e.g.,fermentation vessels would need to be of extraordinary size, and moreimportantly, the likelihood of having all of the planned polypeptidesequence variations represented in the prepared library would decreasesharply. It is therefore preferred to create a designed or biasedlibrary, in which the amino acid positions designated for variation areconsidered so as to maximize the effect of substitution on the RAT orRAT mutant regulation characteristics of the analog, and the amino acidresidues allowed or planned for use in substitutions are limited.

The use of replicable genetic packages, such as the bacteriophages, isone method of generating novel polypeptide entities that regulate RAT orRAT mutant expression. This method generally consists of introducing anovel, exogenous DNA segments into the genome of a bacteriophage (orother amplifiable genetic package) so that the polypeptide encoded bythe non-native DNA appears on the surface of the phage. When theinserted DNA contains sequence diversity, then each recipient phagedisplays one variant of the template (parental) amino acid sequenceencoded by the DNA, and the phage population (library) displays a vastnumber of different but related amino acid sequences.

Such techniques make it possible not only to screen a large number ofpotential binding molecules but make it practical to repeat thebinding/elution cycles and to build secondary, biased libraries forscreening analog-displaying packages that meet initial criteria.

It is well-known to those normally skilled in the art that it ispossible to replace peptides with peptidomimetics. Peptidomimetics aregenerally preferable as therapeutic agents to peptides owing to theirenhanced bioavailability and relative lack of attack from proteolyticenzymes. Accordingly, the present invention also providespeptidomimetics and other lead compounds which can be identified basedon data obtained from structural analysis of the RAT or the RAT mutant.A potential analog may be examined through the use of computer modelingusing a docking program such as GRAM, DOCK, or AUTODOCK. This procedurecan include computer fitting of potential analogs. Computer programs canalso be employed to estimate the attraction, repulsion, and sterichindrance of an analog to a potential binding site. Generally thetighter the fit (e.g., the lower the steric hindrance, and/or thegreater the attractive force) the more potent the potential drug will besince these properties are consistent with a tighter binding constant.Furthermore, the more specificity in the design of a potential drug themore likely that the drug will not interfere with other properties ofthe RAT expression system. This will minimize potential side-effects dueto unwanted interactions with other proteins.

Initially a potential analog could be obtained by screening a randompeptide library produced by a recombinant bacteriophage, for example, ora chemical library. An analog ligand selected in this manner could bethen be systematically modified by computer modeling programs until oneor more promising potential ligands are identified.

Such computer modeling allows the selection of a finite number ofrational chemical modifications, as opposed to the countless number ofessentially random chemical modifications that could be made, and ofwhich any one might lead to a useful drug. Thus, the three-dimensionalstructure and computer modeling, a large number of compounds may berapidly screened and a few likely candidates may be determined withoutthe laborious synthesis of untold numbers of compounds.

Once a potential peptidomimetic or lead compound is identified it can beeither selected from a library of chemicals commercially available frommost large chemical companies including Merck, GlaxoWelcome, BristolMeyers Squib, Monsanto/Searle, Eli Lilly, Novartis and Pharmacia UpJohn,or alternatively the potential ligand is synthesized de novo. Asmentioned above, the de novo synthesis of one or even a relatively smallgroup of specific compounds is reasonable in the art of drug design.

Agents of the present invention may comprise antibodies against the RATpolypeptide or RAT mutant polypeptide. Antibodies against the RATpolypeptide or RAT mutant polypeptide can facilitate selective deliveryof a cytotoxic agent to S. aureus or other bacteria. Alternatively,antibodies can serve as the agent, binding to the RAT polypeptide or RATmutant polypeptide thereby inhibiting activity. The RAT polypeptides orRAT mutant polypeptide, or epitope bearing fragments thereof can be usedas immunogens to produce antibodies immunospecific for suchpolypeptides. Various techniques well known in the art can be usedroutinely to produce antibodies (Kohler, G. and Milstein, C., Nature1975: 256: 495–497; Kozbor et al., Immunology Today, 1983: 4: 72; Coleet al., Monoclonal Antibodies and Cancer Therapy, 1985: pp 77–96).

Accordingly, the present invention also relates to agents identified asinhibitors of RAT gene or RAT mutant gene expression or RAT polypeptideor RAT mutant polypeptide activity and methods for using these agents toincrease lysis of S. aureus and other bacteria, thereby inhibiting theirgrowth and infectivity. These agents can be incorporated into apharmaceutical composition and administered to a host to inhibit growthand infectivity of S. aureus and other bacteria in the host. The term“host” as used herein includes humans.

Pharmaceutical compositions of the present invention comprise aneffective amount of an agent which inhibits the expression of the RATgene or RAT mutant gene or an activity of the RAT polypeptide or RATmutant polypeptide and a pharmaceutically acceptable vehicle. By“effective amount” it is meant an amount which inactivates the RAT geneor RAT mutant gene locus and renders S. aureus or other bacteriasusceptible to killing through cell lysis. The pharmaceuticalcompositions can be administered to a host, preferably a human, toinhibit the growth of S. aureus or other bacteria in the host. Thepharmaceutical composition can be administered alone, or in combinationwith an antibiotic such as penicillin, gentamicin and cephalothin toenhance killing or lysis of the bacteria. Pharmaceutical compositions ofthe present invention can be administered by various routes, including,but not limited to, topically, intramuscularly, intraperitoneally,intranasally, orally, subcutaneously, or intravenously.

The present invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the agents or pharmaceutical compositions of theinvention. The kit may be used for identifying the presence of RAT or aRAT mutant gene or RAT polypeptide or RAT mutant polypeptide in abiological sample by analyzing the sample for the presence of RAT or aRAT mutant. Detection of RAT or RAT mutant cells in a sample areindicative of the patient being susceptible to treatment for thebacterial infection using conventional antibiotic treatment, such aspenicillin. Associated with such container(s) can be a notice in theform of prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, which noticereflects approval by the agency or manufacture, use or sale for humanadministration. In addition, the polypeptides of the present inventionmay be employed in conjunction with other therapeutic compounds.

The embodiments herein described are not meant to be limiting to theinvention. Those of skill in the art will appreciate the invention maybe practiced by using numerous chemical entities and by numerous methodsall within the breadth of the following claims.

1. An isolated RAT polypeptide comprising the amino acid sequence of SEQ ID NO:2. 